c ebpβ specific antibody  (Cell Signaling Technology Inc)

Journal: Immunology and cell biology

Article Title: Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis

doi: 10.1038/icb.2016.63

Figure Lengend Snippet: ( A ) Protein binding in total Gr1 + CD11b + cell population. Gr1 + CD11b + cells were isolated and pooled (n = 6 mice per group) from the bone marrow of sham and septic mice. Cells were fixed in 1% formaldehyde to cross-link protein-DNA complexes. Cells were lyzed and the pelleted nuclei were digested with chromatin shearing enzymatic cocktail. The chromatin solution was then immunoprecipitated with antibodies specific to p-Stat3 (Tyr 705 ), C/EBPβ, C/EBPα, p-Rb (Ser 780 ), or IgG isotype control antibody. Next, chromatin cross-links were reversed to recover the protein-bound DNA. The purified DNA was amplified by qPCR to measure the level of enrichment of miR-21 and miR-181b sequences in the immunoprecipitated complexes using promoter-specific primer/probe sets. PCR reactions were performed in triplicate. Samples were normalized to the “input” DNA (i.e., DNA isolated before immunoprecipitation) and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. (* p ≤ 0.05) of three experiments. *, compare with early sepsis. ( B ) Stat3 and C/EBPβ binding to the miR-21 and miR-181 promoters in Gr1 + CD11b + MDSCs is restriced to the CD31 + subset. The CD31 + cell were then purified from the total Gr1 + CD11b + cell population by positive selection. Cells were treated, and chromatin was immunoprecipitated as described above. PCR was performed to measure the levels of the miR-21 and miR-181b promoter DNA sequence enrichment in chromatin immunoprecipitated from the CD31 + -enriched Gr1 + CD11b + cells from the bone marrow and spleens. Data are expressed as mean ± s.d. of three experiment s .

Article Snippet: The remaining chromatin solution was immunoprecipitated overnight at 4°C with protein G magnetic beads and 3 μg of antibody specific to p-Stat3, C/EBPα, C/EBPβ, p-Rb, or isotype control antibody (Santa Cruz Biotechnology).

Techniques: Protein Binding, Isolation, Immunoprecipitation, Purification, Amplification, Binding Assay, Selection, Sequencing

Journal: Immunology and cell biology

Article Title: Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis

doi: 10.1038/icb.2016.63

Figure Lengend Snippet: (A) Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice and levels of Rb and p-Rb proteins were determined by immunoblot. ( B and C ) Knockdown of cyclin D1 or CDK4 inhibits Rb phosphorylation. CD31 + cells were purified from total Gr1 + CD11b + MDSCs isolated from the bone marrow of late septic mice. Cells were transfected with cyclin D1-, CDK4-specific or control siRNA. After 36 hr, cells were harvested and cell lysates were prepared for immunoblot. ( B ) Levels of the cyclin D1 and CDK4 proteins after the knockdown. Lower panel shows densitometry of the cyclin D1 and CDK4 protein bands. Sample values were normalized to β-actin levels. ( C ) levels of p-Stat3, C/EPBβ, C/EBPα, and p-Rb proteins after cyclin D1 or CDK4 knockdown. The results are representative of three experiments. ( D ) Co-immunoprecipitation analysis of protein binding to Rb. Bone marrow Gr1 + CD11b + cell lysates were prepared and immunoprecipitated with Rb-specific or IgG isotype control antibody using protein G-agarose beads. The immunoprecipitated protein complexes were resolved on denaturing polyacrylamide gel and then immunoblotted with specific antibody against Rb, p-Rb (Ser 780 ), p-Stat3 (Tyr 705 ), C/EBPβ or C/EBPα. ( E ) Co-immunoprecipitation analysis of protein binding to Stat3. Cell lysates were immunoprecipitated with Stat3-specific antibody and immunoblotted as in D . The results are representative of three experiments. ( F ) Protein levels in the crude (input) extract before the immunoprecipitation. The results are representative of two experiments.

Article Snippet: The remaining chromatin solution was immunoprecipitated overnight at 4°C with protein G magnetic beads and 3 μg of antibody specific to p-Stat3, C/EBPα, C/EBPβ, p-Rb, or isotype control antibody (Santa Cruz Biotechnology).

Techniques: Isolation, Western Blot, Purification, Transfection, Immunoprecipitation, Protein Binding

Journal: Immunology and cell biology

Article Title: Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis

doi: 10.1038/icb.2016.63

Figure Lengend Snippet: Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice and transfected with cyclin D1-, CDK4-, Rb-specific or control siRNA for 36 hr. ( A ) Knockdown of cyclin D1 or CDK4 inhibits Rb phosphorylation. Levels of Rb and p-Rb in cell lysate were determined by immunoblot. Cells were fixed, lyzed, and chromatin immunoprecipitation (ChIP) was performed with antibody specific to p-Stat3, C/EBPβ or C/EBPα, as described in . ( B and D ) the recovered, ChIP DNA was amplified by semi-quantitative PCR using primers that flank the Stat3 and C/EBP binding sites in the miR-21 and miR-181b promoters. An IgG-immunoprecipitated samples is shown as a negative control. The results are representative of three experiments. ( C and E ) the recovered DNA was amplified by real-time PCR. Samples values were normalized to the “input” DNA values and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. of three experiments. (F) Levels of c-Myc protein after the Rb knockdown in sepsis Gr1 + CD11b + cells isolated from the bone marrow. The results are representative of two experiments.

Article Snippet: The remaining chromatin solution was immunoprecipitated overnight at 4°C with protein G magnetic beads and 3 μg of antibody specific to p-Stat3, C/EBPα, C/EBPβ, p-Rb, or isotype control antibody (Santa Cruz Biotechnology).

Techniques: Isolation, Transfection, Western Blot, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Immunoprecipitation, Negative Control

Journal: Immunology and cell biology

Article Title: Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis

doi: 10.1038/icb.2016.63

Figure Lengend Snippet: Under normal conditions (i.e., steady-state myelopoiesis), Rb protein is de-phosphorylated via cyclin-dependent kinase (cdk) inhibitor p21-dependent feedback mechanism and thus cannot interact with the C/EBPα protein, allowing it to bind and inactivate the miR-21 and miR-181b promoters. During sepsis, Rb is phosphorylated by a cyclin D1-cdk4 protein complex and interacts with and displaces the C/EBPα protein, thus allowing the Stat3 and C/EBPβ proteins to bind and activate the miR-21 and miR-181b promoters. This differential regulatory pathway requires an IL-6-mediated signal.

Article Snippet: The remaining chromatin solution was immunoprecipitated overnight at 4°C with protein G magnetic beads and 3 μg of antibody specific to p-Stat3, C/EBPα, C/EBPβ, p-Rb, or isotype control antibody (Santa Cruz Biotechnology).

Techniques: